Chromogenic enzyme substrates or chromogenic substrate mixtures are of wide use and importance in the fields of biology and medicine. Body enzymes are detected and measured in clinical chemistry as part of diagnosis of metabolic and other diseases. Enzymes are utilized as a label in biological assays and assay kits, which are based on receptor and analyte pairs (e.g. see U.S. Pat. Nos. 4,193,983 and 4,391,904). In such instances the chromogenic substrate system generates the signal of the assay.
In some cases the biological system or the design of the assay warrant the generation of an insoluble colored product as the signal. The most frequently used examples of such instances will be histochemistry, dot assays, Western blots, and assay kits based on immunoconcentration devices (U.S. Pat. No. 4,632,901).
One of the most frequently used enzymes in the field of biological assays is alkaline phosphatase (Voller, A. and Bidwell, D. "Enzyme-Linked Immunosorbent Assay", in: Rose, N. R., Friedman, H. and Fahey, J. L. (eds) "Manual of Clinical Laboratory Immunology", 3rd Edition, American Society of Microbiology, Washington, D.C., 1986, pp: 99-109). This enzyme has a multitude of substrates, which produce an insoluble, colored product. Some of these substrates and the method for their utilization are presented in the book "Enzyme Histochemistry - A Laboratory Manual" by Z. Lojda, R. Gossrau and T. H. Schiebler, Springer-Verlag, Berlin Heidelberg New York, 1979. Of special interest is the tetrazolium dye method, which involves 2 reaction steps, both occur in the same solution:
1. Primary reaction (enzyme specific)
Indoxyl phosphate ##STR1## Indoxyl+Disodium hydrogen phosphate
2. Secondary reaction (reduction of tetrazolium)
Tetrazolium chloride+Indoxyl.fwdarw.Formazan dye+Indigo white+HCl
The formazan dye precipitates at the location of the reaction. This method is rapid, sensitive and the formed dye is stable.
For preparation of the reaction mixture the indoxyl phosphate and the tetrazolium salt are dissolved in an appropriate solvent, such as dimethyl formamide and then incorporated in a Tris-(tris(hydroxymethyl)aminomethane) or veronal buffer having a pH of 9-10. The mixture thus formed is not stable and will deteriorate within a few hours by spontaneous precipitation of the formazan dye.